Automatic large-scale screening of cells secreting monoclonal antibodies

ABSTRACT

The invention relates to an automatic method for the large-scale in vitro screening of cells, secreting at least one specific monoclonal antibody with affinity for a compound of interest, comprising: (10) distribution of antibody-producing cells on at least one culture plate, (12) culturing of said cells, (14) iterative screening of said cells for the secretion of antibodies with cloning of the cells secreting at least one antibody interacting with the compound of interest and (16) selection of at least one cell secreting a specific monoclonal antibody with affinity for said compound of interest.

The present invention relates to the field of the identification andselection of monoclonal antibodies having advantageous properties inparticular in terms of specificity and affinity. The invention relatesmore specifically to the improvement of the procedures and theproduction of effective means for the purpose of screening suchantibodies.

The subject-matter of the present invention is an automated method forthe large-scale in vitro screening of cells secreting at least onespecific monoclonal antibody with affinity for a compound of interest,said antibody being particularly useful for research, diagnostic and/ortherapeutic purposes.

The present application also describes a method for improving theproduction, by an animal, of cells producing antibodies directed againsta compound of interest, by stimulating the immune response of the animaland by increasing the number of different antibodies produced by saidcells and directed against the compound of interest.

Antibodies belong to the immunoglobulin family. They are produced by theB lymphocytes (or B cells or alternatively “antibody-producing cells”).

Antibodies represent an active means essential for the defense of a hostorganism against a foreign compound. Such a compound may be for examplea parasite, a virus, a bacterium, a polypeptide, a polysaccharide andthe like.

In accordance with usage, the foreign compound referred to above is an“antigen” capable of triggering an immune response in the host organism.More specifically, each antigen comprises one or more “epitopes”,consisting of the specific part(s) of the latter, which react with theantibody or antibodies.

The antigen and the antibody bind according to a “ligand-receptor” typereaction. More particularly, the antibody has, at its surface, a sitecalled “paratope”, capable of recognizing the antigen and correspondingto the site for specific binding with an epitope of this antigen.

In response to the presence of an antigen, the body's immune systemreacts by producing as many different types of antibody as there areepitopes in the antigen.

Dendritic cells are also called “sentinel cells” or “antigen-presentingcells”. These cells allow the stimulation of the humoral and cellularimmune system.

The process consisting in capturing the infectious or tumor antigens,the degradation of the latter in the dendritic cells and thepresentation of the epitopes by these cells are known (Dendritic cells:biology and clinical applications. Ed. M. T. Lotze, A. W. Thomson,Academic Press, 1999). Briefly, the infectious or tumor agents arerecognized and degraded inside the dendritic cells. Next, the, fragmentsthus obtained are presented at the surface of the cells. These foreignfragments are then recognized by the lymphocytes. The humoral andcellular immune process thus solicited leads to the production ofantibodies directed against these tumors and/or these infectious agents.

The recruitment of dendritic cells and the epitope-presenting processare essential steps in the immune response.

The immune response induced by the presence of an antigen comprises thesimultaneous production, by a polyclonal population of B lymphocytes(i.e. a heterogeneous population composed of several types of Blymphocyte), of a multitude of antibodies. In this regard, this immuneresponse is polyclonal in that it results from the production of severaltypes of antibody, each type of antibody being produced by one type of Blymphocyte.

However, the use of polyclonal antibodies for research or diagnosticpurposes, and especially in the context of prophylactic and/ortherapeutic treatments, obviously poses problems of specificity toward aparticular antigen.

These problems have up until now been largely resolved by virtue of thework by Köhler and Milstein, published in 1975 (Nature 256: 495-497).They indeed developed a technique for producing monoclonal antibodies,that is to say antibodies that are homogeneous and of definedspecificity. By virtue of this method and the molecular biologicaltechniques which have now become conventional, the preparation oftailor-made monoclonal antibodies, in unlimited quantities, is ofconsiderable interest in clinical medicine, industry and scientificresearch.

At the present time, it is theoretically possible to design monoclonalantibodies against any type of substance, which was previously difficultto envisage. In general, monoclonal antibodies, by virtue of theirspecificity and their affinity toward particular antigens, constituteidentification tools having very vast fields of application analytical,cytological, histological, functional or biochemical studies. Monoclonalantibodies are thus very widely used in diagnosis and in medical andpharmaceutical research. They also find novel uses in therapy.

Biotechnology companies and laboratories have up until now adoptedvarious approaches and techniques in order to identify the “best”monoclonal antibody, i.e. the monoclonal antibody which is the mostspecific and which has the highest affinity for an antigen involved inthe etiology of a given disease. The best antibody thus identified couldtherefore be advantageously used in the context of the diagnosis,prevention and/or treatment of this disease. However, researchers andengineers do not create an antibody in the same way as a chemicalmolecule could be synthesized. In fact, their work essentially consistsin determining, among the 10¹¹ types of antibody produced by the immunesystem of humans or animals, the antibodies which best correspond to theantigens in question. The strategy is therefore based on theidentification and selection of the best monoclonal antibody orantibodies among a group of potential candidates. This is what iscommonly called “screening”.

Genomic companies specialized in the development of monoclonalantibodies have antibody libraries which are potentially candidates forcombating a large number of diseases. In order to reduce the number ofpotential candidates, all the antibodies of the library are brought intocontact with an antigen. Next, the antibodies which exhibit the highestaffinity for this antigen are selected. However, the libraries consist,not of complete antibodies, but of antibody fragments (so-called phagedisplay technology). Thus, most often, these companies succeed inrapidly identifying antibody fragments of interest. They encounter, onthe other hand, great difficulties as regards obtaining a completeantibody. Indeed, the libraries of antibody fragments are generally notrepresentative of the entire 10¹¹ types of distinct antibodies which theimmune system of an organism is capable of producing. Accordingly, it isnecessary to reconstitute, by engineering techniques, completeantibodies from selected antibody fragments, which may result in theconstruction of antibodies of insufficient specificity and/or affinity.

Cell biology companies have adopted a different approach. Antigens,individualized or not, are injected into animals (humanized transgenicmice or rats). Next, by conventional somatic hybridization, but usinghumanized transgenic mice or rats, a cellular complex is identified,said complex comprising, inter alia, the antibodies which were producedby the mouse or the rat in the presence of the injected antigens.However, these companies do not have the capacities and means necessaryfor, carrying out a rapid and inexpensive screening which makes itpossible to identify each antibody of interest produced by the immunesystem. Indeed, insofar as this approach does not involve techniquescapable of being used on a large scale, it is necessarily limited inthat all the antibodies which are potentially of interest cannot beidentified by a single screening. Consequently, it is likely that thecandidates thus identified are finally not the best in terms ofspecificity and/or affinity.

In addition, it is currently impossible, according to either of theseapproaches, to study in detail the epitopes to which the selectedantibodies bind, without these epitopes being identified beforehand.

In fact, it is currently absolutely essential to have procedures andmeans allowing: on the one hand, the identification of the bestantibodies in terms of specificity and/or affinity, and on the otherhand, the identification of the epitopes recognized by these antibodies.

The method which is the subject-matter of the present invention, whereinmost of the steps and, in any case, wherein all the essential steps, canbe automated, satisfies such concerns for acuity of screening (selectingthe best antibody or antibodies for a given application and, whereappropriate, identifying the epitopes in question), rationalization andsavings, in that it: (i) allows the identification and the selection ofthe best antibody or antibodies among a large number of potentialcandidates; (ii) makes it possible to carry out epitope mapping; (iii)is quick to carry out; (iv) provides reproducible results; (v) makes itpossible to simultaneously screen a large number of antibody-producingcells; (vi) can be carried out by nonspecialist staff; and (vii) can becarried out for routine screening purposes in order to meet the needs ofthe research or analytical laboratory, hospital structure or industry.

For that, the method according to the invention advantageously combineslarge-scale cellular screening and proteomic technology making itpossible to precisely identify the antigen and/or the epitope(s)recognized by a given antibody.

By virtue of this recognition, the invention now allows optimumscreening of antibodies by the identification of the best antibody orantibodies for a given application. Thus, the statistical relevance ofscreening is improved insofar as the method which is the subject-matterof the present invention not only increases the quantity of differentmonoclonal antibodies secreted by the cells subjected to the screening,but also the quality of these antibodies, in terms of specificity and/oraffinity.

The subject-matter of the present invention is therefore an automatedmethod for the large-scale in vitro screening of cells secreting atleast one specific monoclonal antibody with affinity for a compound ofinterest.

The expression “automated” should be understood to mean that all theessential steps of the method in accordance with the invention may be,and are preferably, advantageously automated. In addition, unlessotherwise stated in the remainder of the text, the steps of theparticular embodiments of the method according to the invention may be,and are preferably, also automated.

According, to a first embodiment illustrated by FIG. 1, this methodcomprises at least the following steps:

-   -   (10) distribution of antibody-producing cells in at least one        well of at least one culture plate;    -   (12) culturing said cells (plate culture) under conditions        allowing their growth, with concomitant detection of cellular        growth and of the quality of the cultures;    -   (14) iterative screening of said cells for the secretion of        antibodies, with cloning of the cells secreting at least one        antibody interacting with said compound of interest; and    -   (16) selection of at least one cell secreting one specific        monoclonal antibody with affinity for said compound of interest.

For the purposes of the invention, a “compound of interest” is anantigen comprising at least one epitope. Such a compound is chosen inparticular from: proteins, nucleic acids, viral particles, syntheticpeptides, chemical compounds, organs, organelles (for example Golgiapparatus, mitochondria, and the like), whole cells (for examplemammalian cells, plant cells, bacteria and the like), subcellularfragmentations, (for example fragments of cellular membranes, ofmitochondria and the like). In particular, the abovementioned compoundis a tumor cell. In such a case, the method in accordance with thepresent invention will be advantageously used on the tumor cell ofinterest and, in parallel, on a normal cell derived from the same tissue(normal cell corresponding to the tumor cell).

The “culture plates”, “screening plates” (used during step (14) ofiterative screening indicated above), and “storage plates” forconstituting cell libraries, are as conventionally used for cellcultures. In particular, such a plate comprises 6, 12, 24, 96 or 384wells. Preferably, it comprises 96 or 384 wells.

The culture plates obtained during the abovementioned step (10)represent, in the context of the invention, the “master culture plates”.

Preferably, the distribution of the cells according to step (10) iscarried out in an amount of at least 3×10⁵ cells per well.

The “plate culture” of the cells in accordance with step (12) is carriedout in a conventional selective medium (for example: RPMI medium, 1%mixture of penicillin/streptomycin, 1% pyruvate, 2% glutamine, 10% fetalcalf serum, 1% 8-azaserine., 1% hypoxanthine), generally over a periodof between at least 7 days and at most 21 days, said period beingpreferably between 7 and 15 days. In practice, this period depends onthe density of the cells in the wells. Once the culture period haselapsed, the culture medium is changed automatically. Thus, the oldmedium is collected and used in the context of the iterative screening[step (14)], while the new culture medium is added to the wells of themaster plates.

The “detection of cell growth” concomitantly with the culture [step(12)] may be carried out manually by an operator. In this case, astatistical test may be used. The following estimators may be used.

-   -   Estimator c: “the well is contaminated”, with p(c) distributed        according to a binomial law B(n,p), where n is the number of        wells observed by a qualified operator, and p the probability        for a well to be contaminated; c takes the discrete value 1 if        the well is contaminated, or 0 if nothing abnormal is observed.        According to the maximum likelihood theorem, the test is 95%        reliable if n=30. The operator therefore observes 30 different        wells randomly chosen as follows. The wells are numbered from 1        to N, with 1, the number for the first well of the first 96-well        culture plate (coordinates on the plate: Al), and N the number        for the last well of the last 96-well culture plate (coordinates        on the plate: H12). The numbers for the wells to be observed are        randomly chosen from the list by means of a random number        generator (MS Excel).    -   Estimator C: “at least one well observed is contaminated”, with        p(C) distributed according to a binomial law B(1,P), where P,        the probability for 30 wells to be contaminated, takes the        discrete value 1 if at least one well is contaminated, or 0 if        nothing abnormal is observed. If P(C)=0, it is considered that        all the culture wells are free of contamination with a 95%        reliability. The mean cell growth is empirically evaluated by        the operator on the 30 wells observed.

Alternatively and preferably, this detection is automated, for exampleby means of at least one technique chosen from:

-   -   calorimeter analysis of the pH of the culture medium;    -   measurement of the pH of the culture medium with the aid at        least one probe;    -   analysis of the image of the wells of the plates;    -   measurement of the conductivity of the culture medium by means        of a microelectrode;    -   turbidimetry; or    -   any combination of these techniques.

These are conventional techniques known to a person skilled in the art.

For the purposes of the invention, the “quality of the cultures” refersto the absence of contamination of said cultures.

According to a second embodiment illustrated by FIG. 2, step (14) ofiterative screening comprises at least the following screening module:

-   -   transfer of the culture medium collected from at least one well        of at least one culture plate (#n), into at least one well of at        least one screening plate (#n);    -   screening of the cells for at least one given selection        criterion;    -   selective subculturing of the cells satisfying said criterion        into at least one well of at least one new culture plate (#n+1);        and    -   culture of said cells (“plate culture”) under conditions        allowing their growth, with concomitant detection of cell growth        and of the quality of the cultures.

The expression “module” is understood here to mean a succession of stepswhich can be repeated several times, in a loop. Thus, from one module tothe next, the following will change:

-   -   the initial culture plates (#n), from which the culture medium        (also called here “culture supernatant”) is removed and        transferred;    -   the screening plates (#n);    -   the selection criterion or criteria; and    -   the culture plates (#n+1).

For the purposes of the present invention, the selection criterion ischosen from the following criteria (see FIG. 2): the secretion ofantibodies (“prescreening”); the secretion of antibodies interactingwith the compound of interest (“primary screening”); the secretion ofmonoclonal antibodies specific for said compound of interest (“secondaryscreening”) and the secretion of specific monoclonal antibodies withaffinity for said compound of interest (“tertiary screening”).Preferably, all these criteria are successively applied in the orderindicated above. Under these conditions, the abovementioned module isrepeated four times. It is nevertheless possible to omit theprescreening criterion. The primary screening is then carried outstraight away, reducing the number of repeats of the above module tothree.

Advantageously, when the primary screening is carried out, whether it ispreceded by the prescreening or not, an additional cell cloning step isperformed, as detailed below [step (146); see also FIG. 2].

In addition, the final module performed, corresponding according to theinvention to the tertiary screening, does not necessarily comprise thesteps of selective subculturing Rand culturing on plates. Indeed, theactual tertiary screening is preferably followed by at least one step ofselection of at least one cell secreting a monoclonal antibody whosespecificity and/or affinity for the compound of interest are higher thanthose of monoclonal antibodies secreted by the other cells [step (16),see FIGS. 1 and 2].

The expression “selective subculturing of the cells” corresponds to theusual meaning in the field of cell biology.

In principle, in the context of the iterative screening [step (14)], theplate cultures are performed in standard medium (for example: RPMImedium, 1% mixture of penicillin/streptomycin, 1% pyruvate, 2%glutamine, 10% fetal calf serum). The culture times for step (14) areidentical to the periods indicated above in the case of step (12).

The “detection of cell growth” is as defined above for step (12).

Firstly, the optional prescreening module comprises at least thefollowing steps:

-   -   (140) transfer of the culture medium collected from at least one        master culture plate (at the end of step (12)], to at least one        screening plate (screening plate #0);    -   (141) prescreening of the cells for the secretion of antibodies;    -   (142) selective subculturing of the cells on at least one        culture plate (culture plate #1); and    -   (143) plate culture of said cells.

Step (141) corresponds to a qualitative screening, comprising at least:

-   -   (1411) the detection of the secretion of antibodies; and    -   (1412) the selection of cells secreting at least one antibody.

More specifically, step (1411) of detection of the secretion ofantibodies comprises at least:

-   -   (14111) the collection of at least one culture supernatant        sample; and    -   (14112) the detection of the secretion of antibodies in this        sample.

Alternatively, this step (1411) comprises at least the detection of thesecretion of antibodies directly in the wells.

The prescreening according to step (141), in particular step (1411)above, can be carried out with the aid of any system for detecting a“ligand-receptor” type linkage, known to a person skilled in the art. Byway of examples, there may be mentioned immunodetection systems whichuse isotopes or enzymes, techniques based on the detection ofluminescence or fluorescence, methods using microprobes, and the like.In particular, persons skilled in the art have available conventionalELISA (for Enzyme-linked ImmunoSorbent Assay) techniques, “TopCount” or“Alpha Screen” systems (Perkin Elmer Life Sciences Inc., Boston, Mass.,United States), or FMAT 8100 or FMAT 8200. systems (Applied Biosystems,Manchester, Great Britain). The use of prescreening may involve inparticular conventional micro- or nanotechnology-based means. The samplevolumes required are in this case advantageously less than 10 μl.

Secondly, the primary screening module comprises at least the followingsteps:

-   -   (144) transfer of the culture medium, collected from at least        one culture plate #1 if the prescreening has been carried out,        or from at least one master culture plate in the opposite case,        to at least one screening plate (screening plate #1);    -   (145) primary screening of the cells for the secretion of at        least one antibody interacting with the compound of interest;    -   (146) cloning of the cells secreting at least one antibody        interacting with said compound of interest;    -   (147) subculturing of the cloned cells on at least one culture        plate (culture plate #2); and    -   (148) plate culture of said cells.

The detection of an interaction in each of the wells of a screeningplate, in accordance with the primary screening step (145), isqualitative. This step (145) comprises at least:

-   -   (1451) the collection of at least one culture supernatant        sample;    -   (1452) the detection, in this sample, of the interaction of the        antibodies with the compound of interest; and    -   (1453) the selection of cells secreting at least one antibody        interacting with said compound of interest.

The primary screening referred to in step (145) may be carried out withthe aid of the same techniques as those cited for the prescreening. Hereagain, the primary screening may involve micro- or nanotechnology-basedmeans. Thus, the sample volumes are advantageously less than 10 μl.

The cloning which is the subject of step (146) is aimed at passing frompolyclonal cell populations present in the wells of the prescreeningand/or primary screening plates, to monoclonal cell populations. Such acloning may be carried out by conventional methods well known to personsskilled in the art. For example, there may be mentioned cell sorting byflow cytometry, limiting dilution, performed on a culture plate(generally 96 wells) in standard medium, and cloning in agar, medium. Atthe end of step (146), cells secreting a monoclonal and monospecificantibody are therefore present in each well.

Thirdly, the secondary screening module comprises at least the followingsteps:

-   -   (149) transfer of the culture medium collected from at least one        culture plate #2, to at least one screening plate (screening        plate #2);    -   (150) secondary screening of the cells for the secretion of a        monoclonal antibody specific for the compound of interest;    -   (151) selective subculturing of the cells on at least one        culture plate (culture plate #3); and    -   (152) plate culture of said cells.

The secondary screening step (150) corresponds again to a qualitativescreening. To this effect, this step (150) comprises at least:

-   -   (1501) the collection of at least one culture supernatant        sample;    -   (1502) the detection, in this sample, of a specific interaction        between a monoclonal antibody and the compound of interest; and    -   (1503) the selection of cells secreting a monoclonal antibody        specific for said compound of interest.

This secondary screening may be performed with the aid of the techniquesindicated above for the prescreening and primary screening. In aparticularly advantageous manner, the sample volumes necessary are lessthan 10 μl.

The notion of “specificity” should be understood here in relation to aparticular epitope of the compound of interest. Two scenarios may beenvisaged.

-   -   (i) The compound of interest is a tumor cell. A differential        secondary screening is performed in this case, that is to say        that the results obtained from the qualitative detection of the        “monoclonal antibody—normal cell of the same tissue” linkage        compared with the “monoclonal antibody—tumor cell” linkage are        compared. It is considered in this case that the “monoclonal        antibody—tumor: cell” linkage satisfies the criterion of        specificity if it is detected, whereas a significantly weaker,        or even nonexistent, “monoclonal antibody—normal cell of the        same tissue” linkage is detected.    -   (ii) The compound of interest is different from a tumor cell. It        is appropriate, in this case, to carry out mapping of the        epitope(s), and then to qualitatively determine if the        monoclonal antibody binds to a particular epitope. There is said        to be “specific binding” between the antibody and this epitope        if the latter condition is fulfilled.

If the compound of interest is a tumor cell, persons skilled in the artmay, for a better result, combine the differential secondary screening[case (i)] with epitope mapping [case (ii)].

Fourthly, the tertiary screening module, which is the final module here,comprises at least the following steps:

-   -   (153) transfer of the culture medium collected from at least one        culture plate #3, to at least one screening plate (screening        plate #3); and    -   (154) tertiary screening of the cells for the secretion of a        specific monoclonal antibody with affinity for said compound of        interest.

Optionally, this module additionally comprises:

-   -   (155) selective subculturing of the cells on at least one        culture plate (culture plate #4); and    -   (156) plate culture of said cells.

The tertiary screening which is the subject of step (154) makes itpossible to quantitatively compare the affinity and the specificity ofthe monoclonal antibodies and, where appropriate, to establish a map ofthe epitopes carried by the compound of interest. Thus, step (154)comprises at least:

-   -   (1541) the collection of at least one culture supernatant        sample; and    -   (1542) the measurement of the affinity of a monoclonal antibody        for the compound of interest.

More specifically, the above step (1542) comprises at least:

-   -   (15421) the measurement of the affinity of a monoclonal antibody        for the compound of interest; and    -   (15422) the identification and/or the location of at least one        epitope of said compound of interest.

The identification and/or the location of epitopes (epitope mapping) arealso designated here by the expression “epitope mapping”.

Advantageously, the abovementioned steps (15421) and (15422) may beconcomitant.

Advantageously, the tertiary screening step (154) additionallycomprises:

-   -   (1543) the classification of the monoclonal antibodies on the        basis of their specificity and/or their affinity for the        compound of interest.

The antigen-antibody complex is obtained by spatial complementarity andvia the establishment of bonds of low energy (such as hydrogen,electrostatic, Van der Waals or hydrophobic bonds) between the twoparatopes and the epitope. The sum of these interactions represents amore or less strong overall interaction depending the specificity of theantibody for the epitope. In fact, the antigen-antibody combination isreversible, with an interaction energy which varies from oneepitope-paratope pair to another.

In a 1:1 kinetic model, or monovalent model, the interaction energies ofeach of the two paratopes are not taken into consideration. The overallinteraction energy (namely the energy of dissociation of the complex) isgreater than the arithmetic sum of the interaction energies of the twoparatopes. This overall energy is characterized by an equilibriumconstant, called “affinity constant” or “association constant” K_(D),expressed in L/mol or M⁻¹, and by two kinetic association anddissociation constants, k_(a) and k_(d) respectively (expressed in M⁻¹or min⁻¹). Physically, K_(D) represents the inverse of the minimumantibody concentration necessary for the reaction of formation of thecomplex to be at equilibrium for a given antigen. The affinity constantK_(D) is given by the following formula:

K _(D) =k _(a) /k _(d) =[Ab−Ag]/([Ab]·[Ag])where: [Ab]=antibody concentration

-   -   [Ag]=antigen concentration    -   [Ab−Ag]=concentration of antigen-antibody complex.

In the 2:1 kinetic model, or bivalent model, which is closer to reality,the kinetic association and dissociation constants of each paratope areconsidered: k_(a1), k_(d1) for one site, and k_(a2) and k_(d2) for theother site. The calculation of an overall constant K_(D) is impossiblein this case. Such a model takes into account the fact that theinteraction with one site determines the interaction with the othersite, depending on the accessibility of the epitope. The followingreactions are called into play in this case:

where: Ab₁: first site of interaction of the antibody (or paratope No.1).

-   -   Ab₂: second site of interaction of the antibody (or paratope No.        2).

The constants k_(a1), k_(d1), k_(a2), k_(d2) may then be graphicallymeasured by linearization of standard curves.

In practice, the tertiary screening may be performed in particular bymeans of a device of the Biacore 3000 or Biacore S51 type (Biacore AB,Paris, France). These devices allow the measurement of the kinetics ofinteraction of an antibody with an antigen, the associated constants foreach of the models described above, and the R₅₀, namely the signalobtained for 50% of bound antibodies. To do this, they use themeasurement of the variation of energy of the cloud of free electrons ofa metal (or plasmon) when it is excited by a beam of polarized light.According to one mode of experimentation, the antibodies are bound to ametal plate subjected to laser radiation. An antigen solution is theninjected and “passes” over the bound antibodies with a known flow rate,for a specific period. The apparatus then measures the variations inplasmon energy as a function of the binding of the antigen. Thecalculation of the constants is carried out automatically with the aidof the experimental results thus obtained.

Alternatively, the measurement of the binding affinity [step (15421)mentioned above], represented by the constants K_(D), R₅₀, k_(a), k_(d)in the case of the 1:1 kinetic model, or R₅₀, k_(a1), k_(d1), k_(a2),k_(d2) in the case of the 2:1. kinetic model, and the epitope mapping[abovementioned step (15422)] are carried out by means of at least onetechnique for kinetic analysis of ligand-receptor interaction such as:

-   -   an RIA test,    -   an ELISA test;    -   conventional proteomic techniques known to a person skilled in        the art; and    -   the use of microprobes.

More particularly, in order to map the epitopes, a conventionalproteomic platform is used. Such a platform is generally composed of a2-dimensional electrophoretic system, or 2D electrophoretic system,which makes it possible to cause proteins or protein fragments tomigrate according to two parameters: their molecular weight and theircharge. For example, to analyze the electrophoretic profile of a tumorcell in relation to a normal cell of the same tissue, samples of thesecells are prepared in order to separate the protein fraction. Next, thesamples are placed on a gel and migrate under the effect of an electricfield. During visualization, it is possible to compare, either with theeye, or with the aid of a scanner and a software for image analysis, theelectrophoretic profiles of the two cells, and to identify the signalsor “spots” specific to the tumor cell, or the proteins overexpressed orunderexpressed by the tumor cell compared with the normal cell. Thespots specific to the tumor cells are then manually collected (cut outfrom the gel), or by means of a sampling robot whose operation is linkedto computer analysis of images. The proteins entrapped in the gelfragments are redissolved in solution. Their sequence is determinedusing a Maldi-Toff type mass spectrometer. It is then possible to carryout a new 2D electrophoresis of the peptide fragments identified fromthe results thus obtained on the proteins, and, to map the epitopes ofthe entire protein by adding the screened antibodies to the gel. If thecompound of interest is a protein, only this second 2D electrophoresisis performed, as an alternative to the epitope mapping performed using aBiacore type device (see above). The method with this device is similarto that described above: the antibodies “pass” through a lawn of peptidefragments (a single type of fragments per “lawn”) and interact withdifferent affinities with the latter. This method has the advantage ofproviding, in addition to the mapping, the affinity of the interaction.However, it is more tedious to carry out than 2D electrophoresis.

According to a third embodiment, a cell library is prepared for at leastone screening module of step (14), from the culture plates obtainedafter selective subculturing. Thus, a library is prepared at the end of:

-   -   step (142) for the prescreening module, if the latter is used;        and/or    -   step (147) for the primary screening module; and/or    -   step (151) for the secondary screening module; and/or    -   where appropriate, step (155) for the tertiary screening module.

Preferably, a cell library is prepared for each of the screening modulescited above.

To do this, the cells subcultured on at least one culture plate [steps(142), (147), (151) and (155)] are again subcultured on at least onestorage plate, where they are again cultured over a period of about 7days. Next, the cells are collected, cultured and frozen according totechniques known to persons skilled in the art.

According to a fourth embodiment of the method which is the subject ofthe present invention, illustrated by FIGS. 1 and 2, step (14) ofiterative screening is followed by at least:

-   -   (16) the selection of at least one cell secreting a monoclonal        antibody with specificity and/or affinity for the compound of        interest greater than those of the monoclonal antibodies        secreted by the other cells.

It is possible for this step (16) not to be automated.

According to a fifth embodiment illustrated by FIG. 3, the distributionstep (10) mentioned above is at least preceded by the followingpreliminary steps:

-   -   (1) immunization of at least one animal, with the compound of        interest;    -   (2) optionally, measurement of the immune response of said        animal; and    -   (3) recovery of the antibody-producing cells.

Alternatively, at least the following preliminary steps precede thedistribution step (10):

-   -   (0) bringing at least one dendritic cell (or “antigen-presenting        cell”) and the compound of interest into contact, such that said        dendritic cell presents at least one epitope of said compound of        interest;    -   (1) immunization of at least one animal, with said dendritic        cell presenting said epitope;    -   (2) optionally, measurement of the immune response of said        animal; and    -   (3) recovery of the antibody-producing cells.

At the end of step (0) of bringing into contact, the dendritic cell hasinternalized and cut the compound of interest so as to present fragmentsof said compound at its surface, these fragments comprising, whereappropriate, one or more epitopes of the compound in question.

Preferably, when the compound of interest is a tumor cell, step (0)above comprises at least:

-   -   (01) the fusion of the dendritic cell and the tumor cell; and    -   (02) the recovery of at least one hybrid dendritic cell.

The expression “hybrid dendritic cell” is understood to mean a dendriticcell fused with a tumor cell. Such a hybrid cell advantageously presentsat its surface not only the epitopes normally presented by the tumorcell, but also the cryptic epitopes, which are not normally presented bythe tumor cell. Such a cell, as example II-1 below shows, has theadvantage of possessing the characteristics of the dendritic cell and ofpresenting more epitopes of the tumor cell than the latter, under normalcircumstances.

During the immunization step (1), various adjuvants may be used, inparticular complete or incomplete Freund's adjuvant, or any othermixture of proteins and glcyolipids known to persons skilled in the artfor its use as immunity adjuvant in humans and/or animals.

Several inoculation routes may be envisaged. In particular, thesubcutaneous, intradermal, intravenous, intraperitoneal and intrasplenicroutes, and the like, may be mentioned.

The inoculation may be performed with the aid of a single compound ofinterest or a combination of such compounds, according to programs oftime intervals and quantities of compound(s) of interest which may vary.

The immunization techniques in order to produce antibodies form part ofthe general knowledge of persons skilled in the art.

The above optional step (2) corresponds to the measurement of thehumoral immune response of the immunized animal. This step (2) may beadvantageously carried out with the aid of conventional methods. Forexample, a blood sample is collected from the animal. The circulating Gimmunoglobulins specific for the compound of interest are then assayedby ELISA.

The recovery of the antibody-producing cells according to step (3)consists, for example, in sacrificing the animal in order to collect itsspleen. The cells are then dissociated in the usual manner.

By way of examples of antibody-producing cells suitable for carrying outthe method which is the subject of the present invention, there may bementioned mouse, rat, rabbit and human spleen cells. Preferably, theantibody-producing cells are mouse cells.

According to an advantageous, embodiment, the above preliminary stepsadditionally comprise (see FIG. 3):

-   -   (4) the fusion of the antibody-producing cells thus recovered        with immortalized cells; and    -   (5) the recovery of the immortalized antibody-producing cells.

To do this, preferably, the antibody-producing cells are fused, by theoperator, with tumor, in particular myeloma, cells.

According to a particular embodiment, the preliminary steps mentionedabove [(1), (2), (3) and, where appropriate, (4), (5)] or [(0), (1),(2), (3) and, where appropriate, (4), (5)] are not automated.

According to a sixth embodiment, each step of the method which is thesubject-matter of the present invention is performed in a sterileatmosphere. By default, all the steps of the iterative screening modules[step (14)] may be carried out in a nonsterile atmosphere, as long asthe initial step of each module, corresponding to the transfer of theculture medium to screening plates [steps (140), (144), (149) and(153)], is carried out in a sterile medium. Thus, the addition of thereagents to the wells of the screening plates, the incubation of saidplates and the reading of the results of the screening may be carriedout in a nonsterile atmosphere, even if the compound of interest is acell. In any case, all the steps of the method which is thesubject-matter of the invention which involve antibody-producing cellsare carried out in a sterile atmosphere: in particular, the plateculture steps (12), (143), (148), 152); the transfer steps (140), (144),(149), (153); the selective subculturing steps (142), (147), (151),(155); and the preparation of the cell libraries.

A device for carrying out a method as described above comprises at leastone automatic system controlling:

-   -   at least one part: for controlling at least one robot; and    -   at least one part for data acquisition and processing.

For the purposes of the invention, an “automatic system” is a deviceproviding an automatic and controlled sequence of tasks in accordancewith the instructions of an operator.

Thus, through its operation, this automatic system tends to cancel thedeviation between a controlled parameter (parameter generated by the“automatic control” means, that is to say by the automatic systemitself) and a control parameter (here, the instruction generated by theoperator).

According to the usual meaning, a “robot” is an automatic device capableof handling objects and executing operations according to a fixed orparameterizable program.

A “program” is a sequence of instructions, said “instructions” beingorders expressed in a programming language whose interpretation resultsin the execution of specific elementary operations.

A “parameter” is a variable whose value, address or name is onlyspecified during the execution of the program.

The “data” represent here the results of observations or experiments.

According to particular embodiments, said automatic system is programmedand/or parameterized by the operator (operator instructions).

The automatic system advantageously comprises in this case a memory inwhich at least one program and/or at least one parameter is recorded.

The device in question is such that the automatic system generates theinstructions necessary for carrying out the automated steps and substepsof the method described above.

In this device, the control part of at least one robot controls itself,in accordance with the instructions generated by the automated machine,said robot.

In particular, such a robot is capable of:

-   -   seizing; moving and positioning in (x,y,z) at least one culture,        or screening, or storage plate; and/or    -   storing said plate; and/or    -   collecting liquid medium from at least one well located at a        predetermined position (x,y,z) of said plate; and/or    -   washing said well.

The data acquisition and processing part, present in this device,analyzes, in accordance with the instructions generated by the automaticsystem, the data provided by at least one means for the qualitativeand/or quantitative detection of the cells present in at least one wellof a culture or screening plate.

A detection means which is particularly suitable for use in the devicein question is chosen in particular from:

-   -   a photometric unit for analyzing said well;    -   a unit for analyzing the image of said well;    -   an autoradiography unit comprising at least one means for        measuring the radioactivity of said well;    -   a cell sorting unit comprising at least one means for separating        the cells; and    -   a Biacore 3000 or Biacore S51 type device (see description        above).

All these detection means involve conventional techniques known topersons skilled in the art.

In such a device, at least the parts which act on the antibody-producingcells themselves are in a sterile atmosphere. Thus, the parts of thedevice using the steps of the iterative screening modules [step (14)]may be located in a nonsterile atmosphere as long as the initial stepfor each module, corresponding to the transfer of the culture medium toscreening plates [steps (140), (144), (149) and (153)], is carried outin a sterile medium.

Moreover, the present application discloses a method for improving theproduction, by an animal, of antibody-producing cells directed against acompound of interest.

In the present context, this method makes it possible to stimulate theanimal's immune response and to increase the number of differentantibodies produced by the cells and directed against the compound ofinterest.

According to a first embodiment, the method described here comprises atleast the following steps:

-   -   (20) bringing at least one dendritic cell into contact with the        compound of interest such that said dendritic cell presents at        least one epitope of the compound of interest;    -   (22) immunizing an animal, with the dendritic cell presenting        said epitope;    -   (24) optionally, measuring the immune response of said animal;        and    -   (26) recovering the antibody-producing cells.

The “compound of interest” referred to here corresponds to thedefinition given above.

When the compound of interest is a tumor cell, the method preferablycomprises at least the following steps:

-   -   (30) fusion of at least one dendritic cell and said tumor cell;    -   (32) recovering at least one hybrid dendritic cell;    -   (34) immunizing an animal, with said hybrid dendritic cell;    -   (36) optionally, measuring the immune response of said animal;        and    -   (38) recovering the antibody-producing cells.

A dendritic cell, or an antigen-presenting cell, suitable for carryingout the method according to the invention is for example a mousedendritic cell.

The bringing into contact referred to in step (20) is carried out in aconventional manner, for example in conventional culture plates.

The fusion according to step (30) involves conventional techniques forpersons skilled in the art.

A “hybrid dendritic cell” as mentioned above corresponds to thedefinition above.

Steps (22), (24) and (26) or (34), (36) and (38) are as defined above.

According to a second embodiment, the method described aboveadditionally comprises at least the following steps:

-   -   (40) fusion of the antibody-producing cells thus recovered with        immortalized cells; and    -   (42) recovering the immortalized antibody-producing cells.

These steps are in conformity with the definitions given above.

In addition, the present application discloses the application of themethod described above, for improving the production, by an animal, ofantibody-producing cells, to the large-scale in vitro screening of cellssecreting at least one specific monoclonal antibody with affinity for acompound of interest, in accordance with the method which is the subjectof the invention.

The present invention is illustrated, without however being limited, bythe following figures:

FIG. 1: schematic representation of the essentially automated method forscreening cells secreting at least one monoclonal antibody—overall view.

FIG. 2: schematic representation of the steps relating to the iterativescreening according to step (14) of FIG. 1—detailed view. The dottedarrows indicate the optional steps.

FIG. 3: schematic representation of the preliminary steps relating tothe method schematically presented in FIG. 1. Routes A and B arealternatives.

The examples which follow are intended to illustrate, withoutlimitation, specific embodiments of the present invention.

EXAMPLES

I—Method of Screening:

I-1—Preliminary Steps:

These steps are illustrated in FIG. 3.

A—Steps for Immunization and Recovery of the Antibody-Producing Cells[Steps (1) and (3)]:

In the case of the immunization of a mouse with a tumor cell (compoundof interest), the animal is sacrificed after about 60 to 65 days. Theanimal's spleen is then removed. The cells are then dissociatedaccording to a standard protocol known to persons skilled in the art.

B—Steps for Cell Fusion and Recovery of Immortalized Cells [Steps (4)and (5)]:

The dissociated cells are exposed to murine myeloma cells. The fusionoccurs randomly with a yield estimated at 0.001%. The number ofimmortalized antibody-producing cells (or hybrid cells, or hybridomas)thus generated at about 3×10⁸ cells.

I-2—Method of Screening:

These steps are illustrated by FIGS. 1 and 2.

A—Distribution Step (10):

The antibody-producing cells or the hybridomas are automaticallydistributed by a robot forming part of the device for carrying out themethod, into 96-well culture plates in an amount of 100 000 cells perwell approximately, in a selective medium.

A starting batch of 160 master culture plates are for example thusobtained.

B—Plate Culture Step (12):

The master culture plates are automatically removed daily from theincubator and the culture medium is automatically replaced with newmedium.

After 7 days of culture, the culture medium is changed.

This step leads to the iterative screening step (14) (see FIG. 2).

C—Prescreening Module:

a) Transfer Step (140):

The old medium thus collected is deposited into 160 screening plates(screening plates #0) in an amount of 100 μl per well following exactlythe same well topography as that presented by the culture plates. Thesescreening plates are designated by references in relation to the masterplates using a bar code.

b) Prescreening Step (141):

An anti-IgG antibody coupled to a bead is added in an amount of 10 μlper well to the wells of said screening plates #0. Another antibodycoupled to a fluorescent molecule is added in an amount of 10 μl perwell. After 10 minutes of incubation at room temperature, the screeningplates are read by the FMAT 8200 apparatus (Applied Biosystems) [step.(1411) performed, according to this example, directly in the wells].According to this method of detection, the fluorochrome is excited witha laser. The apparatus generates an image of antibody-bead complexesprovided that at least one IgG population is present in the sample. Thespecific signal is optimized relative to the background noise,corresponding to the signal emitted by the dissociated reagents.

Each well is associated with the number of photons emitted per second byall the complexes. The wells containing no antibody are characterized bya signal/background noise ratio close to 1 and are eliminated. Likewise,the wells for which the signals obtained have an intensity of less thana threshold value set as a function of the calibration of the test, arealso eliminated [step (1412)].

c) Subculturing Step (142):

Depending on these results, the robot selectively collects the cellsfrom the wells of the master culture plates if a significant positivesignal has been detected in the corresponding wells of the screeningplates #0.

The wells considered are then combined and divided into two identicalbatches of 4 plates (batches No. 1 and No. 2). These new culture plates(culture plates #1) are designated by references relative to the masterculture plates with a bar code.

d) Plate Culture Step (143):

The cells are then cultured in the same manner, for 7 days according tothe periodic medium changing protocol described above.

D—Primary Screening Module:

a) Transfer Step (144):

After 7 days of culture, the same cells as those which were used for theimmunization [compounds of interest: step (1) above] are automaticallydistributed into 4 screening plates (screening plates #1) in an amountof 100 000 cells per well, for an initial volume of 50 μl/well.

The culture medium for batch No. 1 of hybridoma culture plates (cultureplates #1) is changed. 50 μl of the old medium collected are redepositedinto the 4 screening plates (screening plates #1) inoculated with thecompounds of interest, following exactly the same well topography. Thesescreening plates #1 are designated by references relative to the cultureplates #1 with a bar code.

Concomitantly, batch No. 2 of 4 culture plates #1 is amplified bytransferring the contents of each well into 3 wells of a new batch of 1296-well plates, and cultured for 7 days. Among each triplicate, thewells where the cells exhibit the best growth is selected and the cellsare subcultured in a well of a batch of 12 24-well plates. Finally,after 7 days of culture, the cells of each well are transferred into 288cell culture flasks. After 7 days of culture, the cells are collected,centrifuged and frozen in liquid nitrogen (−173° C.) in order toconstitute a first cell library.

b) Primary Screening Step (145):

10 μl of an anti-IgG antibody coupled to a fluorescent molecule areadded. After 10 minutes of incubation at room temperature, the screeningplates #1 are read by the FMAT 8200 apparatus (step (1452)]. Thefluorochrome is excited with a laser and the apparatus generates animage of the labeled cells provided that at least one specific antibodypopulation is present in the sample. The specific signal isdistinguished relative to the background noise.

Each well is associated with the number of photons emitted per second byall the cells thus labeled. The wells not containing an antibodyspecific for an epitope present at the surface of the target cell arecharacterized by a signal/background noise ratio close to 1 and areeliminated. Likewise, the wells for which the signals obtained have anintensity less than a threshold value set according to the calibrationof the test, are also eliminated [step (1453)].

The others, that is 15 wells, are subcultured in three batches of 1culture plate (batches Nos. 3, 4 and 5).

Batch No. 3 is amplified by transferring the contents of each well into3 wells of a new batch of 45-well plates, and cultured for 7 days. Foreach triplicate, the well where the cells exhibit the best growth isselected, and the cells subcultured in a well of a batch of 1 24-wellplate. Finally, after 7 days of culture, the cells of each well aretransferred into 15 cell culture flasks. After 7 days of culture, thecells are collected, centrifuged and frozen in liquid nitrogen (−173°C.) in order to constitute a second library.

c) Cloning Step (146):

After maintaining the cultures for 7 days, the cells of batch No. 2 aretransferred by the robot to cloning tubes.

An anti-IgG antibody coupled to a fluorochrome [e.g. a cyanine (AmershamBiosciences.)] is automatically added to the wells. It specificallybinds to the immunoglobulins at the surface of the hybridomas providedthat at least one population of cells (or clone) secretes antibodies.

After incubating for 10 minutes, the cells are cloned in a culture platewith the aid of a sorter-analytical flow cytometer, which selectivelydeposits the cells labeled with the antibody in an amount of one cellper well. 7 positive clones are for example thus obtained.

The steps for subculturing the cloned cells on the culture plates #2,[step (147)] and plate culture [step (148)] are identical to thosedescribed above.

E—Secondary Screening Module:

a) Transfer Step (149):

After 7 days of culture, the same cells as those which were used for theimmunization [compounds of interest: step (1)] are automaticallydistributed in 1 screening plate (screening plate #2) in an amount of100 000 cells per well, for an initial volume of 50 μl/well.

Another batch of two screening plates #2 is inoculated with cellsobtained from the same tissue as the target cells, but having a healthyphenotype. These cells are termed “normal” or “controls”.

The culture medium of batch No. 1 of hybridoma culture plates (cultureplates #2) is changed. The old medium is stored. 50 μl of this mediumare deposited in the screening plate #2 inoculated with the targetcells. Another 50 μl of old medium are deposited in the batch inoculatedwith the control cells following exactly the same well topography. Thesescreening plates #2 are designated by references relative to the cultureplates #2 with bar codes.

b) Secondary Screening Step (150):

10 μl of an anti-IgG antibody coupled to a fluorescent molecule areadded to said screening plates #2.

After incubating for 10 minutes at room temperature, the screeningplates #2 are read by the FMAT 8200 apparatus [step (1502)]. Theapparatus generates an image of the labeled cells, provided at least oneantibody population specific for either of the cell types (tumor ornormal) is present in the sample. The specific signal is distinguishedrelative to the background noise.

By comparison between the wells containing the same, supernatant sample,the antibodies specifically directed against the target cells areidentified. This corresponds for example to 3 clones [step (1503)].

Immediately after collecting the culture supernatants, the cell clonesare amplified by transferring each well into 3 wells of a new batch of21-well plate, and cultured for 7 days.

For each triplicate, the well where the cells exhibit the best growth isselected, and the cells are subcultured in a well of a batch of 124-well plate. Finally, after 7 days of culture, the cells of each wellare transferred to 7 cell culture flasks. After 3 to 7 days of culture,the cells are collected, centrifuged and frozen in liquid nitrogen(−173° C.) in order to prepare a third library.

The steps of selective subculturing of the cells on the culture plates#3 [step (151)] and plate culture [step (152)] are identical to thosedescribed above.

F—Tertiary Screening Module:

a) Transfer Step (153):

The culture supernatants for the 3 clones identified during thesecondary screening are collected and deposited in 3 wells of ascreening plate #3.

b) Tertiary Screening Step (154)

The affinity of the antibodies for the antigen is analyzed using aBiacore 3000 type apparatus [step (1542)].

The antibodies are classified according to the affinity and/or kineticconstants obtained [step (1543)]. At this stage, two antibodies forexample remain.

The total antigens of the target cells are isolated by proteomictechniques and immobilized by Western blotting. The two antibodiesselected during the tertiary screening are deposited on the proteinsthus bound. After incubation, an anti-IgG antibody labeled with afluorochrome (for example fluoroscein) is added. The antigen specificfor each antibody is identified by detection of the fluorescence [step(15422)].

II—Method for Improving the Production of Antibody-Producing Cells:

The example which follows illustrates the case where the compound ofinterest considered is a tumor cell [steps (30) to (38)].

II-1—Materials and Methods [Steps (30) and (32)]:

The experiments were carried out in mice, with murine dendritic cells,and murine myeloma cells (SP2/O).

The hybrid dendritic cells (DH cells) obtained by fusion [step (30)]were analyzed. They possess the character of mouse dendritic cells(recognition by antibodies specific for these cells in cytofluorometry).In addition, they have at their surface the epitopes, including thecryptic epitopes, of the tumor cell. The later characteristic isverified after fusion of dendritic cells and myeloma cells SP2/O andobservation by electron microscopy. The presence of intracisternal Aparticles (IAP) was exclusive to the SP2/O cells, which was confirmed byhybridization.

II-2—Immunization Step (34) and Next Steps:

The immunization protocol was as follows. Four groups of four Balb/cmice were treated as follows:

-   -   group A: 4, mice were immunized with SP2/O cells;    -   group B: 4 mice were immunized with irradiated (UV) SP2/O cells;    -   group C: 4 mice were immunized with nonirradiated DH cells; and    -   group D: 4 mice were immunized with irradiated (UV) DH cells.

The mortality was observed over a standard period of one month after thefirst inoculation.

The analysis of the humoral immune response (or immunization level)[step (36)] was carried out with the aid of the ELISA technique. Thetests were carried out using serum samples diluted one thousand fold on96-well plates coated with SP2/O cells, in order to qualitativelyevaluate the presence of antibodies directed against the antigens ofthese cells in the serum of the treated animals.

The scale adopted for the, measurement was the following:

-   -   0 no response    -   + weak response    -   ++ average response    -   +++ high response    -   ++++ very high response.

The results obtained are the following:

-   -   group A: as expected, the mortality due to metastasis (ascites)        was 100%, with an average survival of 10 to 15 days.    -   group. B: there was no mortality and the immune response against        the SP2/O cells was approximately ±, and could range up to +        against fixed SP2/O cells.    -   group C: the results were similar to those observed with group        A, with a high mortality.    -   group D: there was no mortality and the immune response was        between +++ and ++++.

Groups B and D were subjected to, the inoculation of nonirradiated SP2/Ocells (therefore capable of inducing a tumor response with ascites). Ingroup B, the mortality was 100%, whereas in group D, the mice remain ingood health after 4 months.

1. An automated method for the large-scale in vitro screening of cellssecreting at least one specific monoclonal antibody with affinity for acompound of interest, said method comprising at least the followingsteps: (10) distribution of antibody-producing cells in at least onewell of at least one culture plate; (12) culturing said cells underconditions allowing their growth, with concomitant detection of cellulargrowth and of the quality of the cultures; (14) iterative screening ofsaid cells for the secretion of antibodies, with cloning of the cellssecreting at least one antibody interacting with said compound ofinterest; and (16) selection of at least one cell secreting one specificmonoclonal antibody with affinity for said compound of interest.
 2. Themethod as claimed in claim 1, wherein said compound of interest is anantigen comprising at least one epitope.
 3. The method as claimed inclaim 2, wherein said antigen is chosen from the group consisting ofproteins, nucleic acids, viral particles, synthetic peptides, chemicalcompounds, organs, organelles, whole cells, and subcellularfragmentations.
 4. The method as claimed in claim 3, wherein saidantigen is a tumor cell.
 5. The method as claimed in claim 1, whereinsaid distribution according to step (10) is carried out in an amount ofat least 3×10⁵ cells per well.
 6. The method as claimed in claim 1,wherein each step is performed in a sterile atmosphere.
 7. The method asclaimed in claim 1, wherein said step (10) is at least preceded by thefollowing preliminary steps: (1) immunization of at least one animal,with said compound of interest; (2) optionally, measurement of theimmune response of said animal; and (3) recovery of theantibody-producing cells.
 8. The method as claimed in claim 1, whereinsaid step (10) is at least preceded by the following preliminary steps:(0) bringing at least one dendritic cell and said compound of interestinto contact, such that said dendritic cell presents at least oneepitope of said compound of interest; (1) immunization of at least oneanimal, with said dendritic cell presenting said epitope; (2)optionally, measurement of the immune response of said animal; and (3)recovery of the antibody-producing cells.
 9. The method as claimed inclaim 8, wherein, when said compound of interest is a tumor cell, saidstep (0) comprises at least: (01) the fusion of said dendritic cell andsaid tumor cell; and (02) the recovery of at least one hybrid dendriticcell.
 10. The method as claimed in claim 7, wherein said preliminarysteps additionally comprise: (4) the fusion of said antibody-producingcells with immortalized cells; and (5) the recovery of the immortalizedantibody-producing cells.
 11. The method as claimed in claim 7, whereinsaid antibody-producing cells are chosen from the group consisting ofmouse, rat, rabbit, and human cells.
 12. The method as claimed in claim11, wherein said antibody-producing cells are mouse cells.
 13. Themethod as claimed in claims 7, wherein said preliminary steps are notautomated.
 14. The method as claimed in claim 1, wherein said iterativescreening step (14) comprises at least the following screening module,which may be repeated: transfer of the culture medium collected from atleast one well of at least one culture plate, into at least one well ofat least one screening plate; screening of the cells for at least onegiven selection criterion; selective subculturing of the cellssatisfying said criterion into at least one well of at least one newculture plate; and culture of said cells under conditions allowing theirgrowth, with concomitant detection of cell growth and of the quality ofthe cultures.
 15. The method as claimed in claim 14, wherein said step(14) comprises a prescreening module in which said selection criterionis the secretion of antibodies: (140) transfer of the culture medium toat least one well of at least one screening plate; (141) prescreening ofthe cells for the secretion of antibodies; (142) selective subculturingof the cells secreting at least one antibody on at least one cultureplate; and (143) culture of said cells.
 16. The method as claimed inclaim 14, wherein said step (14) comprises a primary screening module inwhich said selection criterion is the secretion of antibodiesinteracting with said compound of interest: (144) transfer of theculture medium to at least one well of at least one screening plate;(145) primary screening of said cells for the secretion of at least oneantibody interacting with said compound of interest; (146) cloning ofthe cells secreting at least one antibody interacting with said compoundof interest; (147) subculturing of the cloned cells on at least oneculture plate; and (148) culture of said cells.
 17. (canceled)
 18. Themethod as claimed in claim 16, wherein said step (14) additionallycomprises a secondary screening module, in which said selectioncriterion is the secretion of monoclonal antibodies specific for saidcompound of interest: (149) transfer of the culture medium to at leastone well of at least one screening plate; (150) secondary screening ofsaid cells for the secretion of a monoclonal antibody specific for saidcompound of interest; (151) selective subculturing of the cellssecreting a monoclonal antibody specific for said compound of intereston at least one culture plate; and (152) culture of said cells.
 19. Themethod as claimed in claim 18, wherein said step (14) additionallycomprises a tertiary screening module, in which said selection criterionis the secretion of specific monoclonal antibodies with affinity forsaid compound of interest: (153) transfer of the culture medium to atleast one well of at least one screening plate; (154) tertiary screeningof said cells for the secretion of a specific monoclonal antibody withaffinity for said compound of interest; and (155) optionally, selectivesubculturing of the cells secreting a specific monoclonal antibody withaffinity for said compound of interest on at least one culture plate;and (156) optionally, culture of said cells.
 20. The method as claimedin claim 1, wherein said step (16) comprises: (16) the selection of atleast one cell secreting a monoclonal antibody with specificity and/oraffinity for said compound of interest greater than those of themonoclonal antibodies secreted by the other cells.
 21. The method asclaimed in claim 1, wherein said culture is carried out over a period ofbetween at least 7 days and at most 21 days.
 22. The method as claimedin claim 14, wherein a cell library is prepared for at least onescreening module.
 23. The method as claimed in claim 15, wherein saidstep (141) comprises at least: (1411) the detection of the secretion ofantibodies; and (1412) the selection of cells secreting at least oneantibody.
 24. The method as claimed in claim 23, wherein said step(1411) comprises at least: (14111) the collection of at least oneculture supernatant sample; and (14112) the detection of the secretionof antibodies in said sample.
 25. The method as claimed in claim 23,wherein said step (1411) comprises at least the detection of thesecretion of antibodies directly in the wells.
 26. The method as claimedin claim 16, wherein said step (145) comprises at least: (1451) thecollection of at least one culture supernatant sample; (1452) thedetection, in said sample, of the interaction of the antibodies withsaid compound of interest; and (1453) the selection of cells secretingat least one antibody interacting with said compound of interest. 27.The method as claimed in claim 18, wherein said step (150) comprises atleast: (1501) the collection of at least one culture supernatant sample;(1502) the detection, in said sample, of a specific interaction betweena monoclonal antibody and said compound of interest; and (1503) theselection of cells secreting a monoclonal antibody specific for saidcompound of interest.
 28. The method as claimed in claim 19, whereinsaid step (154) comprises at least: (1541) the collection of at leastone culture supernatant sample; and (1542) the measurement of theaffinity of a monoclonal antibody for said compound of interest.
 29. Themethod as claimed in claim 28, wherein step (1542) comprises at least:(15421) the measurement of the affinity of a monoclonal antibody forsaid compound of interest; and (15422) the identification and/or thelocation of at least one epitope of said compound of interest.
 30. Themethod as claimed in claim 29, wherein said steps (15421) and (15422)are concomitant.
 31. The method as claimed in claim 28, wherein step(154) additionally comprises: (1543) the classification of themonoclonal antibodies on the basis of their specificity and/or theiraffinity for said compound of interest.
 32. The method as claimed inclaim 8, wherein said preliminary steps additionally comprise: (4) thefusion of said antibody-producing cells with immortalized cells; and (5)the recovery of the immortalized antibody-producing cells.
 33. Themethod as claimed in claim 8, wherein said antibody-producing cells arechosen from the group consisting of mouse, rat, rabbit, and human cells.34. The method as claimed in claim 33, wherein said antibody-producingcells are mouse cells.
 35. The method as claimed in claim 8, whereinsaid preliminary steps are not automated.
 36. The method as claimed inclaim 10, wherein said preliminary steps are not automated.
 37. Themethod as claimed in claim 32, wherein said preliminary steps are notautomated.
 38. The method as claimed in claim 14, wherein said step (14)comprises: (a) a prescreening module in which said selection criterionis the secretion of antibodies: (140) transfer of the culture medium toat least one well of at least one screening plate; (141) prescreening ofthe cells for the secretion of antibodies; (142) selective subculturingof the cells secreting at least one antibody on at least one cultureplate; and (143) culture of said cells; and (b) a primary screeningmodule in which said selection criterion is the secretion of antibodiesinteracting with said compound of interest: (144) transfer of theculture medium to at least one well of at least one screening plate;(145) primary screening of said cells for the secretion of at least oneantibody interacting with said compound of interest; (146) cloning ofthe cells secreting at least one antibody interacting with said compoundof interest; (147) subculturing of the cloned cells on at least oneculture plate; and (148) culture of said cells.
 39. The method asclaimed in claim 38, wherein said step (14) additionally comprises asecondary screening module, in which said selection criterion is thesecretion of monoclonal antibodies specific for said compound ofinterest: (149) transfer of the culture medium to at least one well ofat least one screening plate; (150) secondary screening of said cellsfor the secretion of a monoclonal antibody specific for said compound ofinterest; (151) selective subculturing of the cells secreting amonoclonal antibody specific for said compound of interest on at leastone culture plate; and (152) culture of said cells.
 40. The method asclaimed in claim 39, wherein said step (14) additionally comprises atertiary screening module, in which said selection criterion is thesecretion of specific monoclonal antibodies with affinity for saidcompound of interest: (153) transfer of the culture medium to at leastone well of at least one screening plate; (154) tertiary screening ofsaid cells for the secretion of a specific monoclonal antibody withaffinity for said compound of interest; and (155) optionally, selectivesubculturing of the cells secreting a specific monoclonal antibody withaffinity for said compound of interest on at least one culture plate;and (156) optionally, culture of said cells.
 41. The method as claimedin claim 14, wherein said culture is carried out over a period ofbetween at least 7 days and at most 21 days.
 42. The method as claimedin claim 21, wherein said period is between 7 and 15 days.
 43. Themethod as claimed in claim 41, wherein said period is between 7 and 15days.
 44. The method as claimed in claim 39, wherein said step (150)comprises at least: (1501) the collection of at least one culturesupernatant sample; (1502) the detection, in said sample, of a specificinteraction between a monoclonal antibody and said compound of interest;and (1503) the selection of cells secreting a monoclonal antibodyspecific for said compound of interest.
 45. The method as claimed inclaim 40, wherein said step (154) comprises at least: (1541) thecollection of at least one culture supernatant sample; and (1542) themeasurement of the affinity of a monoclonal antibody for said compoundof interest.
 46. The method as claimed in claim 45, wherein step (1542)comprises at least: (15421) the measurement of the affinity of amonoclonal antibody for said compound of interest; and (15422) theidentification and/or the location of at least one epitope of saidcompound of interest.
 47. The method as claimed in claim 46, whereinsaid steps (15421) and (15422) are concomitant.
 48. The method asclaimed in claim 45, wherein step (154) additionally comprises: (1543)the classification of the monoclonal antibodies on the basis of theirspecificity and/or their affinity for said compound of interest.